A number of agents have been described for labeling nucleic acids, whether probe or target, for facilitating detection of target nucleic acid. Suitable labels may provide signals detectable by fluorescence, radioactivity, colorimetry, X-ray diffraction or absorption, magnetism or enzymatic activity, and include, for example, fluorophores, chromophores, chemiluminescent compounds, radioactive isotopes, enzymes, and ligands having specific binding partners.
Fluorescent dyes are particularly suitable for detecting nucleic acids. The use of fluorescence labeled polynucleotide probes and polynucleotide hybridization assays have been widely reported. These methods typically require purified DNA or RNA samples for labeling. Other uses of fluorescent dye labeling have been in the field of separation and isolation or purification of nucleic acids from complex biological or clinical specimens.
Typical nucleic acid assays comprise several steps that include target isolation, enzymatic amplification, incorporation of reactive or labeled nucleotides during amplification, purification and detection by hybridization onto specific complementary probe(s). The analysis of target/probe hybridization is the key step in this process and requires the use of fluorescent molecules to specifically label the target nucleic acids. These labels are incorporated into the target DNA or RNA either during enzymatic amplification or post-amplification 5-end enzymatic incorporation of a single fluorescently or biotin-labeled dideoxy-nucleotide.
It is essential that the labeling method not perturb base-pairing hybridization critical for preserving assay specificity. Nevertheless, labeling by enzymatic incorporation often leads to interference with the subsequent hybridization detection step, because current fluorescent labels are attached to the base (purine, pyrimidine) portion of the nucleotides, where base-pairing and hybridization occurs. In addition, enzymatic labeling methods make use of additional enzymatic steps, which require precise calibration to achieve a reproducible labeling yield. Moreover, because the enzymes used depend on the target type (DNA or RNA) and sequence, no specific method is universally applicable to all nucleic acids, and sequence perturbation is often observed.
To remedy this, methods of direct labeling have been used with varying degrees of success, including reactive Cy3 or Cy5-NHS esters, dye amidites or iodoacetamido or bromomethyl dyes. In addition, photobiotin, biotin or digoxigenin-dUTP probes and chemiluminescent detection, fluorescent dendrimer labeling pyrene labeling, as well as radiolabeling methods have been described, but all of these methods suffer from either cumbersome procedures, or are unamenable to direct labeling and hybridization assays using a variety of samples and methods.
Systems for labeling DNA or RNA samples through their phosphate groups have been described in the prior art, however, these methods suffer from obvious problems in the preparation of the active labeling compounds, their solubility, stability as well as their reproducibility in labeling applications. Current methods of preparing active diazomethyl derivatives (hydrazine, MnO2) also involve messy and toxic reagents that are not amenable to biological laboratory use.
The present invention alleviates these difficulties by preparing active diazo labeling reagents in situ, from stable precursor molecules derived from a variety of highly fluorescent dyes and other detection labels. In addition, the present invention defines simple and highly effective methods of activation of the precursor molecules using either highly basic or polymer based reagents, that provide a shelf life of more than six months and are convenient for end-user applications. The systems described in the present invention provide a convenient method for use by biologists and clinicians to label or monitor genomic DNA or RNA samples, nucleotides or oligonucleotides for easy detection and quantification using modern instrumentation and detection systems.
We therefore propose a direct labeling methodology that reduces hybridization and sequence specificity effects by direct (terminal and backbone) phosphate labeling.